PROJECT SUMMARY Alzheimer?s disease is a progressive neurodegenerative disorder with no effective treatment. The only FDA-approved medications are cholinesterase inhibitors (AChEIs), developed based on the Alzheimer?s cholinergic hypothesis. However, AChEI therapy is a modest symptomatic treatment, and no improvement has been made in the last 15 years due to lack of understanding of cholinergic transmission in Alzheimer?s patients. Our parent grant aims to develop and validate families of genetically-encoded acetylcholine (ACh) sensors with sensitivity, specificity, signal-to-noise ratio, kinetics and photostability suitable for monitoring ACh signals in vitro and in vivo. While validating our newly generated ACh sensors, our experiments unveiled a few fundamental, yet unexpected properties of central cholinergic transmission, including the firing pattern-dependent release, restricted volume transmission with subcellular precision and ultra-low release probability. These preliminary results immediately provide an explanation for the low efficacy of current Alzheimer?s medications and point out new effective intervention options for Alzheimer?s disease. In this supplemental application, I propose to interrogate cholinergic transmission in both normal and Alzheimer?s brains following one specific aim, that is to visualize normal and Alzheimer?s cholinergic transmissions. Our plan is to interrogate central cholinergic transmission in the juvenile, adult and aged wild type mice (Aim 1a). Moreover, we will investigate central cholinergic transmission in the juvenile, adult and aged SAMP8 mice, a robust model for sporadic Alzheimer?s disease, as well as in familial Alzheimer's disease animal models, including Tg2576 and/or APP/PS1 mice (Aim 1b). We expect these experiments to characterize cholinergic transmission at different ages in normal brains and reveal how cholinergic transmission may be altered in Alzheimer?s brains.